Antibiotic discovery has slowed dramatically, while the occurrence of antibiotic-resistant infections is increasing. New technologies are required in order to revitalize antibiotic discovery programs. Combinatorial biosynthesis-engineering biosynthetic enzymes to produce variants of these complex molecules may become a powerful tool for development of new antibiotics. Dual-function condensation/epimerization (C/E) domains, which are unusual nonribosomal peptide synthetase domains that catalyze both peptide bond formation and epimerization of the donor L-amino acid, could be powerful tools for combinatorial biosynthesis. Unfortunately, a lack of detailed knowledge about C/E domain structure and function currently limits their utility. If this knowledge gap can be bridged, C/E domains may provide an efficient mean to engineer D-amino acids into nonribosomal peptides and, further, into new antibiotics. Without a detailed understanding of how these enzymes function, it will be impossible to engineer them into nonribosomal peptide synthetase systems. Our long-term goal is to understand C/E domain structure and function in order to apply these bi-functional catalysts in a combinatorial biosynthetic approach to generating unnatural derivatives of peptide antibiotics. The objective of this proposal is to generate preliminary data that will enable formulation of a strong central hypothesis about the substrate specificity and catalytic mechanisms of C/E domains to drive an application for funding at the R01 level. The preliminary hypothesis behind this work is that the gain of epimerization activity in C/E domains is the result of a 50-residue insertion at the N-termini of these enzymes. The rationale for the proposed research is that knowledge of the C/E domain structure and key catalytic and substrate-binding residues will be required in order to manipulate these enzymes in combinatorial biosynthetic systems. Ultimately, this work could lead to new technology that would revitalize antibiotic discovery and, thus, have a profound impact on public health. The preliminary hypothesis will be tested by pursuing the following specific aims: 1) Determine structural features of C/E domains that contribute to the gain of epimerase function; and 2) Identify the roles of active site residues in epimerization and condensation catalysis, and in substrate discrimination. In the first aim, the X-ray crystal structures of unliganded and ligand-bound forms of C/E domains from the enduracidin- producing nonribosomal peptide synthetase will be determined. In the second aim, the roles of highly con- served C/E domain residues will be accessed via site-directed mutagenesis and enzymatic activity assays. The contribution of the proposed studies is to lay the ground work to understand the details of C/E domain structure and function that will be necessary to engineer these unique biocatalysts. This research is significant, because it will provide insights into the structures and functions of the enduracidin biosynthetic enzymes that will facilitate future studies at the R01 level focused on engineering these enzymes to produce enduracidin derivatives.